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MOLECULAR PHARMACOLOGY 51:262-268 (1997).

1,5-Benzothiazepine Binding Domain Is Located on the Extracellular Side of the Cardiac L-Type Ca²⁺ Channel

Junko Kurokawa, Satomi Adachi-Akahane, and Taku Nagao

Department of Toxicology and Pharmacology, Faculty of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan

To determine whether 1,5-benzothiazepine Ca²⁺ channel blocker approaches its binding domain within the cardiac L-type Ca²⁺ channel from inside or outside of the membrane, we tested the effects of a novel potent 1,5-benzothiazepine derivative (DTZ323) and its quaternary ammonium derivative (DTZ417) on guinea pig ventricular myocytes by using the whole-cell patch-clamp technique. The extracellular application of DTZ417 suppressed the L-type Ca²⁺ channel currents (I_Ca(L)) with an IC₅₀ value of 1.2 ± 0.02 µM, which was close to the IC₅₀ value of diltiazem (0.63 ± 0.01 µM). The suppression of I_Ca(L) by DTZ417 was voltage and use dependent but lacked tonic block, which allowed us to investigate the onset of the effect on I_Ca(L) by changing the holding potential (HP) from 90 to 50 mV in the presence of DTZ417. DTZ417 did not have significant effects on I_Ca(L) at an HP of 90 mV. At 50 mV, DTZ417 (50 µM) applied from the extracellular side completely suppressed I_Ca(L), whereas it had no effect from the intracellular side. DTZ323 (1 µM) also inhibited I_Ca(L) only from the extracellular side, without any effects by the intracellular application of 10 µM. However, a quaternary phenylalkylamine derivative, D890 (0.1 mM), acted only from the intracellular side. These results suggest that in contrast to the phenylalkylamine binding site, in cardiac myocytes the 1,5-benzothiazepine binding site is accessible from the extracellular side of the L-type Ca²⁺ channel.